Available online at www.sciencedirect.com

www.elsevier.com/locate/brainres

b r a i n r e s e a r c h 1 6 0 9 ( 2 0 1 5 ) 7 2 – 8 1

http://dx.doi.org/10.0006-8993/& 2015 El

nCorrespondenceMonjolinho, 13565-9

E-mail addressemaragno@ufscar.brltakase@ufscar.br (L

Research Report

The effects of acute and chronic administration ofphosphatidylserine on cell proliferation and survivalin the dentate gyrus of adult and middle-aged rats

Heloisa Maragnoa, Patricia Rodellab,c, Josiane da Silva Freitasd,Luiz Fernando Takasea,n

aDepartamento de Morfologia e Patologia, Centro de Ciências Biológicas e da Saúde, Universidade Federal de São Carlos,São Carlos, BrazilbFaculdade de Farmácia, Centro Universitário da Fundação Educacional de Barretos, Barretos, BrazilcFaculdade de Ciências Farmacêuticas de Araraquara, Departamento de Fármacos e Medicamentos, UniversidadeEstadual Paulista Júlio de Mesquita Filho, São Paulo, BrazildFaculdade de Filosofia Ciências e Letras de Ribeirão Preto, Departamento de Física e Matemática, Universidade de SãoPaulo, Brazil

a r t i c l e i n f o

Article history:

Accepted 9 March 2015

Phosphatidylserine (PS) is an acidic phospholipid that is widely used as an alternative and/

or complementary treatment of cognitive impairments. We hypothesize that these

Available online 17 March 2015

Keywords:

Phosphatidylserine

Neurogenesis

BrdU

Ki-67

Object recognition test

Aging

1016/j.brainres.2015.03.01sevier B.V. All rights rese

to: Departamento de Mo05 São Carlos, SP, Brazils:(H. Maragno), pati.rodell. Fernando Takase).

a b s t r a c t

changes may be attributable, at least in part, to alterations in hippocampal neurogenesis.

The aim of the present study was to investigate the effects of acute and chronic PS

administration on hippocampal cell proliferation and survival in adult (5 months old) and

middle-aged (12 months old) male Wistar rats. PS was injected daily (50 mg/kg, i.p.) during

7 days (acute experiment) or 21 days (chronic experiment). To label newly generated cells,

rats received a single BrdU injection (200 mg/kg, i.p.) one day before PS treatment. The

object recognition test was performed, and the rats were perfused. The brains were

removed and processed with immunohistochemistry techniques for Ki-67 (cell prolifera-

tion) and BrdU (cell survival). The acute and chronic regimens were unable to promote

cognitive improvement in either age group in the object recognition test. The analysis of

cell proliferation showed a significant increase in the number of Ki-67-positive cells after

acute and chronic PS administration in both age groups. The analysis of cell survival

showed that acute and chronic PS administration increased the number of BrdU-positive

cells only in adult animals.

& 2015 Elsevier B.V. All rights reserved.

7rved.

rfologia e Patologia, Universidade Federal de São Carlos, Rod Washington Luis, km 235.

a@gmail.com (P. Rodella), josiane.freitas@hotmail.com (J.d. Silva Freitas),

b r a i n r e s e a r c h 1 6 0 9 ( 2 0 1 5 ) 7 2 – 8 1 73

1. Introduction

Phosphatidylserine (PS) is an acidicphospholipid that is widelyused as an alternative and/or complementary treatment ofcognitive impairment resulting from aging process or neurode-generative disorders (e.g., Alzheimer’s). PS is a natural compo-nent of the brain cortex and represents the major phospholipidof brain synaptic membranes (Breckenridge et al., 1972). It playsan important role in the functioning of neuronmembranes, suchas signal transduction, secretory vesicle release, and cell-to-cellcommunication (Nishizuka, 1984; Blokland et al., 1999). Theaccumulation of PS in synaptic membranes can also modifythe metabolism of glucose (Bruni et al., 1976), catecholamines(Toffano et al., 1976) and acetylcholinesterase (Mantovani et al.,1976). These structural and functional changesmay be correlatedwith the cognitive improvement generally observed after treat-ment with PS in both animals and humans.

Scopolamine-induced cognitive impairments were reversedby PS treatment in rats (Zanotti et al., 1986; Vaisman and Pelled,2009) and mice (Claro et al., 1999, 2006). Similar results wereobserved in rats with cognitive deficits induced by reserpine(Alves et al., 2000). PS administration in middle-aged ratsimproved their performance in memory and learning tests,such as passive and active avoidance (Drago et al., 1981; Zanottiet al., 1989) and the Morris water maze (Nunzi et al., 1992).

Crook et al. (1991) administered PS or placebo in 149patients with age-associated cognitive deficits for 12 weeks.At the end of this period, patients presented significantlybetter performance in memory and learning tests whencompared to the control group. The clinical analysis of thesubgroups showed that patients with the worst memorydeficits were more responsive to PS treatment. In anotherstudy, elderly patients with cognitive deficits showed a sig-nificant improvement in performance in memory and learningtests after treatment with PS for 15 weeks when compared tothe respective control group (Vakhapova et al., 2011).

We hypothesize that the cognitive improvements observedafter PS administration may be attributable, at least in part, toalterations in the production of new brain cells (neurogenesis) inthe hippocampus. Although there are no data in the literatureshowing the effects of PS treatment on hippocampal neurogen-esis, some evidence suggests this important correlation. Structuralchanges in neuronal membranes promoted by the accumulation

Adult Rats

Training STM (2 hs) LTM (24 hs)0

102030405060708090

100

% o

f tim

e

Fig. 1 – Effects of acute PS administration on memory retentionaged animals. (n¼5). Values correspond to the percentage (%) of tdifferences (two-way ANOVA) were detected.

of PS can directly stimulate neurogenesis by inhibiting apoptosis(Kim and Hamilton, 2000; Kim et al., 2000),and increasing cellproliferation (He et al., 2009) and survival (Guo et al., 2007; Kimet al., 2012). These changes can also improve the receptors’efficiency or promote the release of neurotransmitters associatedwith stimulation of hippocampal neurogenesis, such as serotonin,dopamine, and acetylcholine (Casamenti et al., 1979; Heron et al.,1980; Chalon et al., 1998; Park et al., 2012). The function of adulthippocampal neurogenesis has been strongly linked to learningand memory (Gould et al., 1999; Shors et al., 2001) and itsdisruption has been hypothesized to be important in the devel-opment and maintenance of several human psychopathologies(Jacobs et al., 2000; Malberg et al., 2000).

The aim of the present study was to investigate the effectsof acute and chronic PS administration on episodic memory,and hippocampal cell proliferation and survival in adult andmiddle-aged rats.

2. Results

2.1. Object recognition test

The object recognition test aims to assess short- and long-term episodic memory. The index of memory retention isrepresented by the percentage of time the animal spendsinteracting with a new object rather than a familiar one.

Acute and chronic regimens were unable to promote cogni-tive improvement in either age group. No significant differencesin memory retention were observed after acute PS administra-tion in the short-term memory test (adult, t5¼0.563, P40.05;middle-aged, t5¼1.145, P40.05) and long-term memory test(adult, t5¼0.127, P40.05; middle-aged, t5¼0.352, P40.05)(Fig. 1). Chronic PS administration presented similar results inthe short-term memory test (adult, t5¼0.6718, P40.05; middle-aged, t5¼1.299, P40.05) and the long-term memory test (adult,t5¼1.973, P40.05; middle-aged, t5¼0.672, P40.05) (Fig. 2).

2.2. Cell proliferation

The effect of acute and chronic PS administration on cellproliferation was assessed by quantitative analysis of thenumber of Ki-67-positive cells in the dentate gyrus of adultand middle-aged rats.

Middle-aged Rats

Training STM (2 hs) LTM (24 hs)0

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100ControlPS

% o

f tim

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in the object recognition test. (A) Adult animals; (B) middle-ime exploring the new object7SE. No statistically significant

Adult Rats

Training STM (2 hs) LTM (24 hs)0

102030405060708090

100

% o

f tim

e

Middle-aged Rats

Training STM (2 hs) LTM (24 hs)0

102030405060708090

100 Control

PS

% o

f tim

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Fig. 2 – Effects of chronic PS administration in memory retention in the object recognition test. (A) Adult animals; (B) middle-aged animals. (n¼5). Values correspond to the percentage (%) of time exploring the new object7SE. No statistically significantdifferences (two-way ANOVA) were detected.

Table 1 – Effect of acute PS administration on the number of Ki-67 positive cells in the dentate gyrusof adult and middle-aged rats.

Region Adult (5 months) Middle-aged (12 months)

Control (n¼5) PS (n¼5) Control (n¼5) PS (n¼n¼5)

Dentate gyrus 30487258.3 48847423.0 nnn (60.2%) 10307160.9 1818754.7 nnn (76.6%)Dorsal 11217134.7 16977166.6 (51.4%) 384787.4 765788.8 n (99.2%)Ventral 19277157.3 31877314.0 nnn (65.4%) 6467114.5 1053737.4 nn (63.1%)

Subgranular zone 19277205.2 27867231.5 n (44.6%) 526776.4 783777.8 (49.0%)Dorsal 742778.9 998756.6 (34.6%) 192748.7 330760.1 (71.9%)Ventral 11867164.9 17887240.9 (50.8%) 334758.6 §§ 453719.2 (25.8%)

Hilus 1121782.5 20987264.9 nn (87.2%) 502793.7 1035727.0 nnn (105.4%)Dorsal 379767.1 6987122.1 (84.2%) 192749.3 435732.3 (þ126.6%)Ventral 742728.7 13997162.0 (88.6%) 310761.9 600755.9 (92.3%)

Values are means7SEM.Statistical analysis: two-way Anova and Bonferroni’s multiple comparison test.n pr0.05 vs control.nn pr0.01 vs control.nnn pr0.001 vs control.

Acute PS - Cell Proliferation

DGDorsa

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6000Control AdultFTS AdultControl Middle-agedPS Middle-aged

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Fig. 3 – Effect of acute PS administration on the number of Ki-67positive cells in the dentate gyrus of adult and middle-aged rats.

b r a i n r e s e a r c h 1 6 0 9 ( 2 0 1 5 ) 7 2 – 8 174

Acute PS administration promoted a significant increase incell proliferation in adult and middle-aged animals (Table 1 andFigs. 3 and 4). In the adult group, the analysis showed that the PSsignificantly increased the number of proliferating cells in thedentate gyrus (60.2%), its ventral portion (65.4%), subgranular

zone (44.6%), and hilus (87.2%). In the middle-aged group, PSpromoted a significant increase inKi-67-positive cells in thedentate gyrus (76.6%), its dorsal and ventral portions (99.2%and 63.1%, respectively), and hilus (105.4%).

Chronic PS administration promoted a significant increase incell proliferation only in adult animals (Table 2 and Fig. 5).Quantitative analysis showed a significant increase in the num-ber of proliferating cells in the dentate gyrus (105.1%), its ventraland dorsal portions (107.0% and 103.0%, respectively), in thesubgranular zone (288.2%), and its ventral and dorsal portions(282.4% and 293.4%, respectively). Despite themarked stimulationin cell proliferation in adult animals, the chronic regimen wasunable to increase cell proliferation in middle-aged rats.

Similar to previous reports (Cowen et al., 2008), we foundthat the number of proliferating cells in the hippocampussignificantly declined with age (Tables 1 and 2). The averagedecline observed in the acute experiment was 62.4%; however,the subgranular zone showed a higher degree of decline(�72.0%) compared to the hilus (�51.4%).

2.3. Cell survival

The effects of acute and chronic PS administration on cellsurvival were assessed by quantitative analysis of the

Table 2 – Effect of chronic PS administration on the number of Ki-67 positive cells in the dentate gyrusof adult and middle-aged rats.

Region Adult (5 months) Middle-aged (12 months)

Control (n¼5) PS (n¼5) Control (n¼5) PS (n¼5)

Dentate gyrus 11867137.8 24317319.6 nnn (þ105.1%) 11707101.4 12637144.4 (7.9%)Dorsal 617785.0 12777168.5 nn (þ107.0%) 435791.7 5167115.0 (18.6%)Ventral 569763.5 11547185.3 n (þ103.0%) 735792.4 747764.1 (1.6%)

Subgranular zone 346752.4 13427163.1 nnn (þ288.2%) 588756.9 495789.4 (�15.8%)Dorsal 182733.6 7187101.6 n (þ293.4%) 207759.9 204757.3 (�1.4%)Ventral 163727.0 6247104.6 n (þ282.4%) 381744.5 291734.4 (�23.6%)

Hilus 840794.4 10907174.3 (þ29.7%) 579768.1 768784.6 (32.6%)Dorsal 434766.0 559783.4 (þ28.7%) 228743.5 312763.1 (36.8%)Ventral 406736.7 5307109.9 (þ30.8%) 351759.5 456762.7 (29.9%)

Values are means7SEM.Statistical analysis: two-way Anova and Bonferroni’s multiple comparison test.n pr0.05 vs control.nn pr0.01 vs control.nnn pr0.001 vs control.

Fig. 4 – Effect of acute PS administration on the number of Ki-67 positive cells in the dentate gyrus of adult and middle-agedrats. Adult control group (A), adult PS group (B), middle-aged control group (C) and middle-aged PS group (D). SGZ, subgranularzone; GCL, granular cell layer. Arrows indicate Ki-67-positive cells. Scale bar¼100 μm.

b r a i n r e s e a r c h 1 6 0 9 ( 2 0 1 5 ) 7 2 – 8 1 75

number of BrdU-positive cells in the dentate gyrus of adultand middle-aged rats.

The analysis of the early stages of cell survival in the acuteexperiment showed that PS administration promoted a sig-nificant increase in cell survival only in the dentate gyrus(24.3%) of adult animals (Table 3 and Fig. 6). No significantdifferences were observed in middle-aged animals.

Chronic PS administration promoted a significant increase incell survival only in adult animals (Table 4). The analysisshowed that the PS significantly increased the number ofproliferating cells in the dentate gyrus (110.3%), its ventralportion (238.7%), in the subgranular zone (153.3%), and its

ventral portion (343.5%). No differences in cell survival wereobserved in the dentate gyrus of middle-aged rats.

3. Discussion

In general, this study shows that despite the administrationof PS not promoting cognitive improvements, a significantincrease in hippocampal cell proliferation and survival wasobserved. The comparison of the results observed in both agegroups also showed that administration of PS was more

Table 3 – Effect of acute PS administration on the number of BrdU positive cells in the dentate gyrusof adult and middle-aged rats.

Region Adult (5 months) Middle-aged (12 months)

Control (n¼5) PS (n¼5) Control (n¼5) PS (n¼5)

Dentate gyrus 19327169.5 24027220.5n (24.3%) 792762.7 864791.8 (9.1%)Dorsal 7877127.7 9047104.2 (14.8%) 276736.6 297758.2 (7.6%)Ventral 1145761.6 14987144.7 (30.9%) 516798.2 567758.9 (9.9%)

Subgranular zone 11647110.2 13987159.4 (20.1%) 388753.2 561763.4 (44.6%)Dorsal 535785.7 586769.6 (9.5%) 16077.9 225737.4 (40.6%)Ventral 629745.9 8127101.1 (29.1%) 228760.7 336770.5 (47.4%)

Hilus 7687100.0 1004790.6 (30.7%) 40479.5 303793.0 (�25.0%)Dorsal 252762.1 318750.8 (26.2%) 116730.7 72734.3 (�37.9%)Ventral 516745.4 686762.6 (32.9%) 288739.6 231768.3 (�19.8%)

Values are means7SEM.Statistical analysis: two-way Anova and Bonferroni’s multiple comparison test.n po0.05 vs control. nn pr0.01 o vs control. nnn pr0.001 o vs control.

Table 4 – Effect of chronic PS administration on the number of BrdU positive cells in the dentate gyrusof adult and middle-aged rats.

Region Adult (5 months) Middle-aged (12 months)

Control (n¼5) PS (n¼5) Control (n¼5) PS (n¼5)

Dentate gyrus 955777.6 2009782.6 nnn (þ110.3%) 792766.8 6027158.3 (�18.3%)Dorsal 5527100.0 643751.3 (þ16.5%) 2767179.2 226772.1 (�27.0%)Ventral 403752.1 1366748.2 nnn (þ238.7%) 5167114.5 3777108.3 (�23.9%)

Subgranular zone 545759.1 1380732.4 nnn (þ153.3%) 388797.1 350774.7 (�9.7%)Dorsal 341744.5 475739.9 (þ39.4%) 160714.4 134732.5 (�16.0%)Ventral 204732.2 905735.1 nnn (þ343.5%) 2287110.9 216749.9 (�5.3%)

Hilus 410744.6 629764.2 (þ53.2%) 404717.4 252785.1 (�37.6%)Dorsal 211759.1 168727.9 (�20.5%) 116756.0 91739.9 (�21.4%)Ventral 199751.3 461752.9 (þ131.3%) 288772.3 161760.4 (�44.2%)

Values are means7SEM.Statistical analysis: two-way Anova and Bonferroni’s multiple comparison test.nnn pr0.001 vs control. nn pr0.01 o vs control. n pr0.05 o vs control.

b r a i n r e s e a r c h 1 6 0 9 ( 2 0 1 5 ) 7 2 – 8 176

effective in promoting changes in adult animals (5 months)than in the middle-aged ones (13 months).

Administration of PS was able to reverse scopolamine-inducedamnesia in rats (Zanotti et al., 1986) and mice (Claro et al., 2006).Middle-aged animals also showed a significant cognitive improve-ment in the Morris water maze after acute administration of PS(Lee et al., 2010; Suzuki et al., 2001). The present study shows thatboth acute and chronic regimens did not promote an improve-ment in memory retention in the object recognition test in adultor middle-aged rats. Despite having a beneficial effect on drug- oraging-induced memory deficits, PS does not seem to have amemory improving effect per se, i.e., in non-treated healthy adultanimals (Claro et al., 1999; Castilho et al., 2004).

The absence of cognitive improvement maybe explained bythe level of differentiation andmaturation of these new neurons.The new cells produced in the subgranular zone must migrate tothe granular cell layer and differentiate into mature neurons toparticipate in the hippocampal circuitry responsible for theacquisition of new memories (Gould et al., 1999; Kempermann,2002). Thus, cognitive improvements in the memory test may beobserved 28 days after PS treatment, the time required formigration, differentiation, and maturation of new neurons.

The object recognition test also assesses episodic recogni-

tion memory (Dere et al., 2005). Perhaps other memory tests,

such as the habituation test in the open field, spatial learning

in the water maze, or aversive memory in the inhibitory

avoidance test, may show different results.At present, there are no data in the literature showing the

effects of PS administration on hippocampal neurogenesis inadult or middle-aged rats; however, several studies suggestthis important correlation. Docosahexaenoic acid (DHA) is anomega-3 fatty acid that is a primary structural component ofthe human brain, comprising 40% of the polyunsaturatedfatty acids in the brain. DHA plays an important role in thedevelopment and functioning of the CNS, both in humans(Birch et al., 2000) and rats (Gamoh et al., 1999). In adultanimals, DHA increases hippocampal neurogenesis by twodifferent mechanisms: promoting cell survival through inhi-bition of apoptosis (Kim and Hamilton, 2000; Kim et al., 2000,2012) and by directly stimulating cell proliferation (He et al.,2009). Diet supplementation with DHA promotes a significantincrease in PS concentration in neuronal membranes. DHApositively modulates PS biosynthesis and accumulation inneurons, promoting cell survival (Guo et al., 2007).

Chronic PS - Cell proliferation

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Control AdultPS AdultControl Middle-agedPS Middle-aged

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Fig. 5 – Effect of chronic PS administration on the number ofKi-67 positive cells in the dentate gyrus of adult and middle-aged rats.

Fig. 6 – Effect of acute PS administration on the number of BrdU positive cells in the dentate gyrus of adult. Adult control group (A)and adult PS group (B). SGZ, subgranular zone; GCL, granular cell layer. Arrows indicate BrdU-positive cells. Scale bar¼100 μm.

b r a i n r e s e a r c h 1 6 0 9 ( 2 0 1 5 ) 7 2 – 8 1 77

The administration of PS promoted a significant increasein hippocampal cell survival. Neuronal apoptosis is consid-ered an important factor in the cell survival mechanism(Gould et al., 1990; Tanapat et al., 1999). Apoptosis occursmainly during development and maturation of the CNS(Sastry and Rao, 2000), but programmed cell death can stillbe observed in the dentate gyrus of adult animals (Biebl et al.,2000). Under normal conditions, about 50% of new cellsproduced in the hippocampus enter apoptosis (Dayer et al.,2003). Kim et al. (2000) demonstrated in vitro that thedecrease of PS in cell membranes is usually associated withthe induction of neuronal apoptosis, which is partiallyreversed by DHA administration. DHA is incorporated intoneuronal membrane phospholipids and modifies their phy-sicochemical properties, promoting signaling targets relatedto membrane cell survival (Kim et al., 2012). DHA alsopromotes an increase of Raf-1 in the cell membrane (Kimand Hamilton, 2000). Raf-1 is essential for the transduction ofseveral growth-factor proto-oncogenes, promoting cell survi-val (Hoyle et al., 2000).

PS can also stimulate cell proliferation through structuraland functional changes in the neuronal synaptic membranes

(Beltz et al., 2007), increasing the fluidity (Yehuda et al., 1998)and improving their efficiency of binding to neurotransmitters,including serotonin (Heron et al., 1980). Serotonin has animportant role in proliferation of new neurons during adult-hood (Jacobs, 2002; Malberg and Duman, 2003) by acting as alocal endogenous factor that stimulates neurogenesis (Jacobs,2002).

Other neurogenesis-related neurotransmitters, such asacetylcholine and dopamine, can be stimulated by PSadministration. PS can control the activation of the choli-nergic system by stimulating the production and release ofacetylcholine in the CNS (Casamenti et al., 1979), and byincreasing the activity of acetyltransferase and the reactivityof acetylcholinesterase in the hippocampus (Park et al.,2012). The hippocampus receives input from several fore-brain cholinergic structures. Acetylcholine is directlyinvolved in the formation and maintenance of short-termmemory, retention processes, and retrieving long-termmemories (Izquierdo and Medina, 1997; Mizoguchi et al.,2001). The cholinergic system may be involved in theregulation of neurogenesis, since newly formed cells in the

hippocampus receive various cholinergic projections (Kotaniet al., 2006) and express nicotinic and muscarinic receptors(Kaneko et al., 2006). Selective activation of these receptorsstimulates cell proliferation in the dentate gyrus and CA1region (Van Kampen and Eckman, 2010). Hippocampal cho-linergic denervation significantly inhibited cell proliferation(Mohapel et al., 2005).

Diet supplementation with fatty acids that compose the cellmembrane, such as PS, decreased MAO-B activity andincreased endogenous levels of dopamine binding sites(Chalon et al., 1998). A high concentration of dopamine rec-eptors was observed in proliferative zones in the brain, such asthe subventricular zone and hippocampus (Diaz et al., 1997).Evidence suggests that dopamine is a potent stimulator ofendogenous neural precursor cell proliferation in the hippo-campus in rodents (Baker et al., 2004; Höglinger et al., 2004) andprimates (Freundlieb et al., 2006). Administration of the selec-tive D2 and D3 receptor agonist quinpirole significantlyincreases hippocampal neurogenesis through ciliary neuro-trophic factor (Yang et al., 2008), an important endogenousregulator that promotes self-renewal or maintenance of neuralprecursors (Chojnacki et al., 2003).

b r a i n r e s e a r c h 1 6 0 9 ( 2 0 1 5 ) 7 2 – 8 178

In the present study, middle-aged animals showed lowerresponsiveness to PS administration when compared to theadult group. Only the acute regimen promoted an increase incell proliferation, whereas neither acute nor chronic treat-ments were able to promote significant changes in cell survivalin the middle-aged group. However, studies in the literatureshowed important physiological changes after PS administra-tion, such as formation of new synapses, dendrites, andmembrane receptors (Wurtman et al., 2009), as well as opti-mization of the release of several neurotransmitters (Uluset al., 1989; Schaechter and Wurtman, 1990).

Morphological and physiological changes in the gastro-intestinal tract caused by aging may explain the lowereffectiveness of PS in middle-aged animals. The small intes-tine presents a significant decrease in the absorption ofnutrients, resulting from the decrease of 40–50% of theintestinal villi and splanchnic blood flow. Another factor isthe reduced absorption of various nutrients such as vitaminD, folic acid, vitamin B12, and fatty acids (Ferrioli et al., 2006).

Another factor that may explain the lower responsivenessto PS in middle-aged animals is the stress caused by dailyintraperitoneal injections. The physiopathology of aging isoften accompanied by a decline in stress tolerance at bothsystemic and cellular levels (Hall et al., 2000). Aged andmiddle-aged animals usually have higher blood levels ofglucocorticoid due to changes in the regulation of thehypothalamic–pituitary–adrenal axis. Elderly rodents presentexacerbated endocrine responses to stress, and despite thepeak of stress-related hormones remaining the same, gluco-corticoids take longer to return to basal levels (Sapolsky et al.,1983), and furthermore, high levels of glucocorticoids signifi-cantly inhibit hippocampal neurogenesis (Gould et al., 1997).These data suggest that new routes of administration, suchas gastric gavage, should be tested in middle-aged animals tooptimize the absorption and reduce the stress caused by theinjections.

The present study showed that acute and chronic PSadministration promoted a significant increase in cell pro-liferation and survival in the dentate gyrus of adult rats. Theresults also showed that the middle-aged group did notrespond as well to the PS treatment as the adult group; onlythe acute PS treatment promoted an increase in cell prolif-eration in this age group.

4. Experimental procedures

4.1. Animals

Adult, male Wistar rats, aged5 and 12–13 months, were grouphoused (3 animals per cage) under controlled lighting (12-hlight/12-h dark cycle, light on at 07:00 h) and temperature(22 1C71 1C) conditions, in standard polycarbonate cages withfree access to food and water. All experiments were conductedin accordance with the guidelines of the National Council forControl of Animal Experimentation (Concea), Brazil, and wereconducted with the approval of the Federal University of SãoCarlos Animal Care and Use Committee. All precautions weretaken to minimize any animal pain or discomfort.

4.2. PS and BrdU administration

Phosphatidylserine (from Fagron, Brazil) was prepared freshdaily. The drug was dissolved in sterile saline (0.9%) andsonicated. To evaluate effects on hippocampal proliferationand cell survival, the PS suspension was injected intraper-itoneally (i.p.) at a dose of 50 mg/kg at light onset for 7 days(acute experiment) or 21 days (chronic experiment). Anequivalent volume of the drug vehicle served as the controlin these experiments. The main fatty acids present in thepreparation are C16:0 (2.7%), C18:0 (39.5%), C18:1 (35.3%),C20:1 (6.1%), C22:1 (6.4%), C24:1 (3.3%), and C22:6 (6.7%), andtheir purity is 92%.

To label newly generated cells in the hippocampus, ratsreceived a single i.p. injection of 5-bromo-20-deoxyuridine (BrdU;200mg/kg), 24 h prior to the initiation of PS or drug-vehicletreatment. The BrdU (Sigma-Aldrich Company, St. Louis, MO)was dissolved in sterile saline (containing 0.007 N NaOH) andgiven in a volume of 10ml/kg body weight. The effect of acuteand chronic PS administration on the subsequent survival ofthe newborn cells was determined at the end of the study.

4.3. Object recognition test

The object recognition test is based on the typical behavior ofratsto spend more time exploring novel objects than familiarones. This preference can be used as an index of memory forobject recognition (Ennaceur and Delacour, 1988; Dere et al.,2005).

The apparatus consisted of an open box (100�100�50cm) made of wood, the inside of which was painted white.Four objects made of glass were used, two identical (A and B)and two different from each other (C and D). The weight ofthe objects ensured that they could not be displaced bythe rats.

The test started 6 days prior to the perfusion day. All ratswere given four habituation sessions (once per day, per-formed at the beginning of the dark period) in which theywere allowed to explore the empty apparatus (without theobjects) for 5 min. On the object trial, the rat was placed intothe box with the two identical objects (A and B) and allowedto explore for 5 min (training). The rat was then removed toits home cage. Two hours after training, the short-termmemory test was performed. The rat was placed again inthe box with two objects, one of which was the same as theone used for training (A—familiar object) and the other was anovel object (C). The rat was allowed to explore the objects for5 min, and the time that the animal explored each object wasmeasured. Twenty-four hours after training, the long-termmemory test was performed. The rat was placed in the box,but object C was changed for another novel object (D) that therat had never encountered before. The rat was allowed toexplore the objects for 5 min, and the time that the animalexplored each object was measured.

Object exploration was deemed to be when the rat direc-ted the nose to the object at a distance of less than 2 cm.Turning around or sitting on the object was not considered asexploratory behavior. The following parameters were ana-lyzed: the percentage (%) of time that the animals exploredeach of the identical objects during training, and the

b r a i n r e s e a r c h 1 6 0 9 ( 2 0 1 5 ) 7 2 – 8 1 79

percentage of time that the animals explored the novel andfamiliar objects in the short-term and long-term memorytests. The percentage of time used for novel object explora-tion was considered as an index of memory retention.

4.4. Perfusion

Rats were deeply anesthetized with ketamine (�150 mg/kg,i.p.) and were transcardially perfused with cold physiologicalsaline, followed by 4% paraformaldehyde in 0.1 M phosphatebuffer. Brains were removed, post-fixed, and cryoprotected,and then sectioned on a freezing microtome.

Frozen coronal sections (40-μm thick) were collectedthroughout the entire hippocampus and separate series oftissue (1-in-12 sections) were then processed for BrdU (cellsurvival) and Ki67 (cell proliferation), using a slide-mountedimmunoperoxidase technique (Fornal et al., 2007).

4.5. Ki-67 and BrdU immunohistochemistry

For BrdU staining, sections were boiled in citric acid, digestedwith trypsin, denatured with hydrochloric acid, and then incu-bated with a mouse monoclonal antibody raised against BrdU(Novocastra Laboratories Ltd, Newcastle upon Tyne, UK) for 48 hat 4 1C. For Ki67 staining, sections were boiled in citric acid, andthen incubated with a mouse monoclonal Ki67 antibody (NCL-Ki-67-MM1; Novocastra Laboratories Ltd) for 48 h at 4 1C. Followingprimary antibody incubation, sections were incubated with abiotinylated horse anti-mouse IgG and with avidin–biotin com-plex (Vector Laboratories, Burlingame, CA), and then reacted with3,30-diaminobenzidine (DAB) to visualize labeled cells. Sectionswere then counterstained with cresyl violet, dehydrated, andcoverslipped. Slides were then coded prior to analysis.

All slides were analyzed blind by an experienced scorer,using an Olympus B202 light microscope. In every 12th section,BrdU-positive and Ki67-positive cells (stained brown) werecounted bilaterally in the DG at 400� magnification. The cellcounts for each animal were summed across all sections andthen multiplied by 12 to obtain an estimate of the total numberof labeled cells in the DG. In addition, the DG was divided intoanterior (dorsal) and posterior (ventral) portions, as in Guzmán-Marín et al. (2003). Briefly, the boundary separating the anteriorand posterior portions of the DG corresponded to the regionwhere the CA2 and CA3 pyramidal cell layers coalesce into acontinuous cell layer in the coronal plane, i.e., �4.5 mm fromthe bregma, according to the atlas of Paxinos and Watson(1986). The number of labeled cells was also counted separatelyin the subgranular zone (SGZ) and in the hilus. Cells locatedwithin two cell-body widths from the border of the granular celllayer were considered to be in the SGZ; cells located moredistally were considered to be in the hilus.

4.6. Statistical analysis

All data are expressed as means7SEM. Statistical analysis ofgroup data was performed using atwo-way analysis of var-iance (ANOVA) followed by post hoc Bonferroni multiplecomparison tests. A probability value (P) o0.05 was taken asstatistically significant.

Acknowledgments

We gratefully acknowledge the generous financial supportfrom FAPESP (process 2007/52445-5) for making this workpossible. H.M. was a recipient of a fellowship from theBrazilian government (CNPq).

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